Neuronal co-transmission, the phenomenon whereby a neuron releases more than one neurotransmitter, is a generalized property of many synapses in the central and peripheral nervous system. Previous work has shown that serotonergic neurons can release both serotonin and glutamate and recent studies have shown that this co-transmission plays a significant role in the generation of behavioral output. However how the use of two neurotransmitters, serotonin and glutamate, uniquely shapes the regulation of postsynaptic targets remains poorly understood. Serotonergic neurons of the Median and Dorsal Raphe nuclei project to the hippocampus where they innervate pyramidal cells and interneurons. Surprisingly the synapses made by serotonergic neurons onto GABAergic interneurons of the SR and SLM appear to rely primarily on glutamate acting on ionotropic receptors, while those made onto pyramidal cells appear to rely primarily on serotonin acting on metabotropic 5-HT1A receptors. This suggests that co-transmission in hippocampus facilitates the differential regulation of distinct postsynaptic targets, a phenomenon that has previously been reported for co-transmission in invertebrates and in autonomic ganglia. In this application we propose to elucidate the mechanisms underlying this remarkable dichotomy. The experiments proposed in this application will be conducted using whole cell electrophysiological recordings in in vitro hippocampal brain slices. Slices will be prepared from SERT-Cre driver mice selectively expressing channelrhodopsin in serotonergic neurons. We and others have previously shown that serotonergic synapses onto GABAergic interneurons of the SR and SLM rely on GluA receptors to elicit robust EPSCs with only sparse evidence for serotonin co-transmission. In the first specific Aim we will examine the potential involvement of different mechanisms that can account for this phenomenon. Additionally we will also test the idea that expression of serotonergic co-transmission is stimulation frequency dependent. In contrast to the situation in interneurons, serotonergic synapses onto pyramidal cells rely primarily on serotonin to elicit 5-HT1A receptor mediated slow IPSCs. In the second Specific Aim we will use intersectional genetic strategies to separate synapses made by neurons co-releasing serotonin and glutamate and test the hypothesis that pyramidal cells are selectively innervated by neurons releasing only serotonin. We will also test the possibility that serotonergic neurons may release glutamate that acts on higher affinity NMDA receptors. The results of these experiments should clarify key mechanistic aspects of serotonin glutamate co-transmission in the hippocampus and contribute to a better understanding of the pathophysiology and therapeutics of diseases involving serotoninergic synapses.